Summary of Work: Currently studies have focused on the role of MutS and its associated ATPase activity in both replication and recombination. MutS belongs to a superfamily of ATPases that contains both the Walker A & B consensus motifs. The exact role of ATP binding/hydrolysis in methyl-directed repair is not known but has stimulated a great deal of interest because of the internal conservation of mismatch repair in higher cells. We now know that nucleotide binding and not hydrolysis is all that is required for loss of mismatch specificity by mutS. Moreover, it has been proposed that there exists two modes for MutS dissociation from the mismatch. These two modes of mismatch release are thoguht to be depedent upon ATP binding/ and subsequent hydrolysis where the latter leading to DNA translocation. To help clarify the biochemistry behind MutS ATPase activity we have carried out site- directed mutagenesis to define the active site residue (carboxylate) responsible for hydrolysis. We have preliminary evidence to suggest aspartic acid 661 is the active site residue. Currently, we are characterizing several other residues (Asp 651, 662, & 673 and Glu 646) that could potentially particpate in activation of a water molecule. Gel-shift assays reveal that only non-conserved changes (Asp or Glu to Ala) perturbed mismatch binding. All conserved changes bound a G/T mismatch almost as efficient as wild-type. Only MutSD651N and MutSD661N exhibited reduced ATPase activity with Kcat values between 1-2. In vitro mismacth repair assays showed that only mutSD661N failed to complement RK 1517 (MutS-) extracts. We are presently testing the mode of ATP dissociation by these mutants to distinquish the dissociation modes. Previously our lab investigated the dominant negative effect associated with two MutS mutants in vitro. We were, however, unable to dissect the genetics behind this dominant negative phenotype in vivo with our given biochemical strategies. Therefore we decided to tackle this problem utilizing an approach that would allow us to prepare heterodimers between mutant and wild type MutS. This procedure involved differentially GST- and his6- fusion proteins. The separate tags facilitated rapid and pure 1:1 mutant-wild-type heterodimers. However, this procedure was met with failure in that the GST-fusion was not active. We therefore constructed a different tag utilizing the affinity of strepavidin for strepavidin binding peptide. Preliminary studies suggest a 1:1 ratio of mutant to wild type hinders repair but does not completely block it. We are currently testing the possibility that MutS bidirectional repair capacity is a function of independent ATP-driven monomer translocation. In examining further roles of mismatch repair in recombination we demonstrate that MutS & MutL proteins block RuvAB- dependent branch migration between M13-fd DNA. This effect was mismatch dependent, as branched intermediates between M13-M13 were unimpeded by these activities. - E. Coli, DNA Repair, DNA Binding Protein, Mutagenesis, Recombinant Proteins